KR101275766B1 - Composition for inhibiting angiotensin converting enzyme, or having antihypertensive or antiobese property, comprising protein extract of abalone intestine - Google Patents

Abstract

The present invention comprises an abalone extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate, and mixtures thereof, as an active ingredient, for inhibiting angiotensin converting enzyme , Antihypertensive or anti-obesity compositions.

Description

Composition for inhibiting angiotensin converting enzyme, or having antihypertensive or antiobese property, containing an abalone viscera protein extract, a hydrolyzate of the extract, a fraction of the hydrolyzate, or a mixture thereof , comprising protein extract of abalone intestine}

The present invention relates to an anti-hypertensive or anti-obesity composition for inhibiting Angiotensin-I Converting Enzyme (ACE) containing an abalone viscera protein extract, a hydrolyzate of the extract, a fraction of the hydrolyzate or a mixture thereof. will be.

Abalone belongs to the gastropoda, also known as bok (鰒) or po (한) in Chinese. It is called "Fugu" in "Jean-Abo", and it is called "Seokmyeong-myeong" in "Herbaceous cortex" and is also known as Gugong-ra (라).

In Korea, the Korean wave abalone (Haliotis discus hannai), the turbulent hakkatis (Haliotis discus), horse abalone (H. gigantea), Sibolt abalone (H. Sieboldii), H. diversicolor diversicolor, Madae five molecular (H) 6 species are known, including diversicolor supertexta).

This abalone is one of the main seafood products that have been used for food since ancient times. Domestic abalone production was only around 100 tons per year until 2002. Most of the production is natural, and in the early 2000s, when farming started in earnest, only 20 tons of aquaculture were produced and 60 to 70 tons were produced. The production of aquaculture has been ahead of natural production since 2002, and by 2003, the production of aquaculture has increased dramatically to 1,065 tons and only 73 tons.

As such, production of aquaculture abalone is expected to increase annually. However, it is the market which is a big problem. The current export trend is mainly sold only in live abalone, and the main export countries are limited to Japan.

In light of the increase in abalone production in Korea, the export market is still not open. These situations can lead to various problems. One of them is the price cut. Price cuts can increase exports for the time being, but they are not an alternative for the long-term overturning industry. Therefore, it is urgent for fishermen to expand the sales channels of abalone and to develop various new exportable products such as secondary processed products and other high value products in addition to live abalone.

On the other hand, due to the deterioration of immune function due to drinking, smoking, high obesity, high blood pressure, cancer and aging, there is an increasing interest in functional food, it is urgent to search and develop functional materials.

Meat, which is the main edible part of abalone, contains a large amount of bioactive substance taurine, and contains bioactive substances such as amino acids, proteins, and EPA (functional unsaturated fatty acids). Abalone, which mainly consumes seaweed such as seaweed and seaweed, contains various enzymes that degrade natural polysaccharides and proteins in seaweed.

However, there are few studies on abalone physiological function so far, so it is urgent to improve the product value of abalone and establish basic data by scientifically and systematically exploring the physiological function and effectiveness of abalone.

In addition, the development of functional food and pharmaceutical ingredients in abalone would increase the added value of abalone and import substitution effect on high value-added products, thus increasing the demand for the development of functional food and pharmaceutical materials for abalone. have.

The inventors of the present invention confirmed that the angiotensin converting enzyme inhibitory effect, antihypertensive and anti-obesity effect of the abalone viscera protein extract, the hydrolyzate of the protein extract, the fraction of the hydrolyzate, or a mixture thereof, among various extracts of various parts of the abalone. It is an object of the present invention to provide angiotensin-converting enzyme inhibitor, antihypertensive, or anti-obesity composition containing these as active ingredients.

An object of the present invention as described above, the composition for inhibiting angiotensin converting enzyme containing an active ingredient selected from the group consisting of abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate and mixtures thereof Is achieved.

It is also an object of the present invention to achieve an antihypertensive composition comprising an abalone protein extract, a hydrolyzate of the protein extract, a fraction of the hydrolyzate, and a mixture thereof, as an active ingredient.

It is also an object of the present invention, containing as an active ingredient selected from the group consisting of abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate and mixtures thereof, It is achieved by an anti-obesity composition.

Preferably, the hydrolyzate may comprise the abalone viscera protein as pepsin, trypsin, chymotrypsin, pancreatin, alcalase, flavozyme, papain It can be obtained by treatment with a proteolytic enzyme selected from the group consisting of (papain) and mixtures thereof.

Preferably, the fraction may be obtained by subjecting the hydrolyzate to liquid chromatography using an eluting solvent selected from the group consisting of buffered solutions, acidic aqueous solutions, water and mixtures thereof.

Preferably, the composition may be a pharmaceutical composition or a food composition.

The present invention can provide a composition exhibiting an anti-hypertensive and anti-obesity effect, including a significant angiotensin converting enzyme inhibitory effect, and can be used as a pharmaceutical or food composition to prevent or treat hypertension, arteriosclerosis or obesity. It is possible to obtain the effect of providing a drug or food for.

Since the active ingredient of the present invention which brings about such an effect is obtained from abalone, the high value of the abalone can be achieved by this invention.

1 is a diagram showing the hypertension mechanism by angiotensin converting enzyme.
Figure 2 is a graph showing the results of measuring the ACE inhibitory activity of the extract of Comparative Examples 1 to 12.
Figure 3 is a graph showing the results of measuring the ACE inhibitory activity of the extracts of Examples 13 and 14.
4 is a graph showing the results of measuring pancreatic lipase inhibitory activity of the extracts of Examples 1 to 12.
5 is a graph showing the results of measuring pancreatic lipase inhibitory activity of the extracts of Examples 13 and 14.
Figure 6 is a graph showing the results of measuring the ACE inhibitory activity of Examples 15 to 18.
7 is a graph showing chromatogram showing peaks of three fractions of Example 19 and ACE inhibitory activity.

The present invention comprises an abalone extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate, and mixtures thereof, as an active ingredient, for inhibiting angiotensin converting enzyme , Antihypertensive or anti-obesity compositions.

Therefore, hereinafter, abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract and a method for producing a fraction of the hydrolyzate, and extracts, hydrolysates and fractions obtained thereby will be described. The following describes angiotensin converting enzyme inhibition, antihypertensive or anti-obesity compositions containing these as active ingredients.

Abalone flesh extract and abalone viscera extract of the present invention can be prepared by the following method.

The abalone flesh extract and abalone are separated from the abalone by separating the flesh and viscera from each abdomen, and extracting each site using conventional solvents according to conventional methods known in the art, i.e., under conditions of normal temperature and pressure. The viscera extract may be prepared, but may be preferably prepared by extracting using a solvent extraction method, a reflux extraction method or an ultrasonic extraction method using an extraction solvent.

Abalone flesh extract and abalone viscera extract of the present invention can be prepared using a solvent extraction method using an extraction solvent in the above extraction method.

Abalone flesh extract and abalone viscera extract in the present invention is a variety of extraction solvents, for example, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, butanol, etc.), propylene glycol, 1,3 -Butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof can be obtained using an extraction solvent selected from the group consisting of It is not.

According to a preferred embodiment, the preparation method of abalone flesh extract and abalone viscera extract of the present invention are as follows.

First, abalone meat and abalone viscera are washed and chopped, and then lyophilized for at least 48 hours and used as a sample while refrigerated at 2 to 8 ° C.

When preparing abalone flesh extract, the extraction solvent of 80 to 250 times the dry weight of abalone flesh is used, and when preparing abalone gut extract, the extraction solvent of 40 to 200 times the dry weight of abalone gut is used, and 100 to 300 rpm After extracting for 10 hours or more using a filter paper and the filtrate was evaporated using a rotary evaporator to extract all the extraction solvent to obtain the abalone flesh extract and abalone viscera extract, respectively.

In addition, the abalone flesh extract and abalone viscera extract of the present invention includes not only the extract by the above-mentioned extraction solvent, but also the extract that has undergone a conventional purification process. Obtained through various additional purification methods, such as, for example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). The active fraction is also included in the abalone flesh extract and abalone viscera extract of the present invention.

In addition, the reflux extraction method is a method to obtain the extract by reflux extraction using a reflux extraction device.

The extraction solvent that can be used for the reflux extraction method is selected from the group consisting of chloroform, ethanol, methanol, ethyl acetate, hexane, diethyl ether and mixtures thereof.

As an example of a method for producing the abalone flesh extract and abalone viscera extract of the present invention by the reflux extraction method, washing and cutting abalone flesh and abalone viscera, respectively, lyophilized for at least 48 hours and refrigerated at 2 to 8 ℃ Use as a sample.

The sample was placed in an extraction solvent selected from chloroform, ethanol, methanol, water, ethyl acetate, hexane, diethyl ether, and mixtures thereof, followed by reflux extraction at 80 to 120 ° C. for at least 1 hour using a reflux extraction apparatus, followed by hydrothermal extract. In the case of using only filter paper to collect the filtrate and freeze-dried, the other organic solvent extract is collected by filtering the filtrate using a rotary evaporator to extract all the extract solvent of the present invention to obtain the abalone flesh extract and abalone viscera extract of the present invention, respectively. The extraction solvent is used in an amount of 20 to 150 times the sample dry weight.

In addition, the ultrasonic extraction method is an extraction method using energy generated by the ultrasonic vibration, the ultrasonic wave can destroy the insoluble solvent contained in the sample in the water-soluble solvent, due to the high local temperature is generated Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained, and the impact efficiency of the ultrasonic energy induces high pressure to increase the mixing effect of the material and the solvent contained in the sample, thereby increasing the extraction efficiency.

The extraction solvent that can be used in the ultrasonic extraction method is selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane, diethyl ether and mixtures thereof.

The extracted sample is vacuum filtered to recover the filtrate, and then the extraction solvent is removed by a reduced pressure evaporator, and the extract may be obtained through a conventional extract preparation method of freeze drying.

As an example of the preparation method of the abalone flesh extract and abalone viscera extract of the present invention by the ultrasonic extraction method, after washing and cutting abalone flesh and abalone viscera, each sample is lyophilized for 48 hours or more and the sample is refrigerated at 2 to 8 ℃ Used as.

The sample is placed in an extraction solvent selected from chloroform, ethanol, methanol, water, ethyl acetate, hexane, diethyl ether, and mixtures thereof, followed by extraction with an ultrasonic extractor at an intensity of 20 to 60 KHz for at least 1 hour. The ultrasonic extraction time may vary depending on the amount of sample to be extracted. The extraction solvent is used in an amount of 20 to 150 times the sample dry weight.

In the case of hot water extract, only the filtrate is collected and freeze-dried using a filter paper, and other organic solvent extracts are collected by filtering the filtrate and evaporating all the extraction solvents using a rotary evaporator to obtain the abalone flesh extract and abalone viscera extract of the present invention. .

Abalone flesh extract and abalone viscera extract of the present invention obtained by such a method has a significant anti-obesity effect, this effect will be described in more detail in the experimental example.

Abalone viscera protein extract of the present invention can be prepared by the following method.

The abalone intestine is washed, cut and freeze-dried for at least 48 hours to prepare a powder form. To the abalone viscera powder, buffer solution, such as 50 mM sodium phosphate buffer solution (pH 7.4), 50 mM potassium phosphate buffer solution (pH 7.0), or 50 mM Tris-HCl (pH 7.5), was added 20 to 200 times the dry weight of the abalone viscera powder. After adding to the amount of, and extracted at 2 to 8 ℃, at least 100 hours at 100 to 300 rpm, centrifuged at 2,000 to 8,000 rpm to obtain a supernatant, 20 to 100% (w / v) ammonium sulfate in the supernatant ) To precipitate the protein, and the protein is dialyzed at 2 to 8 ° C. for at least 24 hours using a dialysis tube and then lyophilized to obtain the abalone viscera protein extract of the present invention.

The abalone viscera protein extract of the present invention obtained by the above method has an angiotensin converting enzyme inhibitory effect and an antihypertensive effect, and also shows a remarkable anti-obesity effect, which will be described in detail in the experimental example.

In addition, the angiotensin converting enzyme inhibitory effect, antihypertensive effect and anti-obesity effect are more prominent in the hydrolyzate of the abalone viscera protein extract and the fraction of the hydrolyzate.

The preparation method of the hydrolyzate of the abalone viscera protein extract of the present invention is as follows.

The abalone visceral protein extract obtained by the above method is a proteinase, pepsin, trypsin, chymotrypsin, pancreatin, alcalase and flavozyme. ), Papain or mixtures thereof are added and reacted at 100 to 300 rpm for at least 24 hours at a temperature of 30 to 60 ° C.

The mixing ratio of the abalone viscera protein extract and proteolytic enzyme is preferably 300: 1 to 10,000: 1 by weight.

The optimum reaction temperature and pH of the above proteolytic enzymes are shown in Table 1 below.

enzyme
Temperature (℃)
pH
pepsin
37
2.0
trypsin
37
7.0
α-chymotrypsin
37
7.0
pancreatin
37
8.0

After the reaction described above, the proteolytic enzyme was inactivated by agitation at 90 to 120 ° C. for 10 to 20 minutes, and then, the supernatant was taken by centrifugation for 5 to 20 minutes at a speed of 9,000 to 15,000 rpm, and the frozen solution was frozen. Drying gives a hydrolyzate of the abalone gut protein extract of the present invention.

Method for producing a fraction of the hydrolyzate of the abalone viscera protein extract of the present invention is as follows.

The hydrolyzate of the abalone viscera protein extract obtained by the above method was subjected to liquid chromatography using an eluting solvent selected from the group consisting of buffer solution, acidic aqueous solution, water, and mixtures thereof, A fraction of the hydrolyzate is obtained.

As the buffer solution, any general buffer solution may be used, and in particular, sodium phosphate buffer solution, potassium phosphate buffer solution or a mixture thereof may be used. Preferably, as the buffer solution, 50mM sodium phosphate buffer solution (pH 7.0), 50mM potassium phosphate buffer solution (pH 7.0), 50mM sodium phosphate buffer solution (pH 8.0) and the like may be used.

As the acidic aqueous solution, an acetic acid aqueous solution, an aqueous hydrochloric acid solution, and the like can be used, and preferably a 10 mM aqueous acetic acid solution (pH 2.0), a 10 mM aqueous hydrochloric acid solution (pH 2.0), or the like can be used.

Preferably, a fraction of the hydrolyzate is obtained by performing liquid chromatography using water as the elution solvent.

More preferably, the fraction of the hydrolyzate is subjected to liquid chromatography using water as the eluting solvent to obtain three fractions and may be the second fraction of these three fractions.

The hydrolyzate of the abalone viscera protein extract of the present invention and the remarkable angiotensin converting enzyme inhibitory effect, antihypertensive effect and anti-obesity effect of the fraction of such hydrolyzate will be described in more detail in the experimental example.

Such an abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fraction of the hydrolyzate or a mixture thereof as an active ingredient, for inhibiting angiotensin converting enzyme, antihypertensive or Referring to the anti-obesity composition is as follows.

Angiotensin converting enzyme inhibitor or antihypertensive composition of the present invention contains the abalone viscera protein extract, the hydrolyzate of the protein extract, the fraction of the hydrolyzate or a mixture thereof as described above as an active ingredient.

The composition of the present invention comprises the abalone viscera protein extract, the hydrolyzate of the protein extract, the fraction of the hydrolyzate or a mixture thereof based on the total weight of the composition of 0.00001 to 20% by weight, preferably 0.0001 to 10 It is contained in the content of 0.00% by weight, more preferably 0.001 to 5% by weight. When the abalone viscera protein extract or the like is contained less than 0.00001% by weight based on the total weight of the composition, the angiotensin converting enzyme inhibitory effect or antihypertensive effect due to the inclusion of the abalone viscera protein extract is hardly expected. When the abalone viscera protein extract and the like is contained in an amount of more than 20% by weight based on the total weight of the composition, there is no apparent increase in effect with increasing content.

The anti-obesity composition of the present invention contains an abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fraction of the hydrolyzate or mixture thereof as an active ingredient as described above.

Such a composition of the present invention, the abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate or a mixture thereof based on the total weight of the composition of 0.00001 to 20 weight %, Preferably 0.0001 to 10% by weight, more preferably 0.001 to 5% by weight. When the abalone flesh extract and the like is contained in an amount of less than 0.00001% by weight based on the total weight of the composition, the anti-obesity effect due to the inclusion of the abalone flesh extract can hardly be expected. If it contains more than 20% by weight based on weight, there is no apparent increase in effect with increasing content.

Such a composition of the present invention may be a pharmaceutical composition or a food composition.

Abalone viscera protein extract of the present invention, hydrolyzate of the protein extract, fraction of the hydrolyzate or mixture thereof as an active ingredient for angiotensin converting enzyme inhibition or antihypertensive pharmaceutical composition, or abalone flesh extract of the present invention, abalone Anti-obesity pharmaceutical compositions containing as an active ingredient viscera extracts, abalone viscera protein extracts, hydrolysates of the protein extracts, fractions of the hydrolysates, or mixtures thereof, are suitable carriers, excipients, and the like commonly used in the manufacture of pharmaceutical compositions; It may further comprise a diluent, and the pharmaceutical dosage forms of the pharmaceutical compositions containing the perennial enzyme treatment extracts, fractions thereof or mixtures thereof, may be used alone or in combination with other pharmacologically active compounds, as well as in suitable collections. .

Abalone viscera protein extract of the present invention, hydrolyzate of the protein extract, fraction of the hydrolyzate or mixture thereof as an active ingredient for angiotensin converting enzyme inhibition or antihypertensive pharmaceutical composition, or abalone flesh extract of the present invention, abalone Anti-obesity pharmaceutical compositions containing viscera extracts, abalone viscera protein extracts, hydrolysates of the protein extracts, fractions of the hydrolysates, or mixtures thereof as active ingredients, respectively, powders, granules, tablets, capsules according to conventional methods Oral formulations such as suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alzenate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrididone, water, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

When formulating the pharmaceutical composition of the present invention, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like commonly used.

Solid preparations for oral administration of the pharmaceutical compositions of the present invention may include tablets, pills, powders, granules, capsules, etc., wherein such solid preparations comprise at least one excipient in the perennial enzyme-treated extract, fractions thereof or mixtures thereof. For example, it can be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral use of the pharmaceutical composition of the present invention include suspensions, solvents, emulsions, syrups, and the like, which include various excipients, for example, wetting agents, in addition to water and liquid paraffin, which are commonly used simple diluents. Sweeteners, fragrances, preservatives and the like can be included.

Formulations for parenteral administration of the pharmaceutical compositions of the invention may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used, and suppository bases such as witepsol and macrogol , Tween 61, cacao butter, laurin, glycerogelatin and the like can be used.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, intravenous, intramuscular or subcutaneous injection.

Abalone viscera protein extract of the present invention, the hydrolyzate of the protein extract, fractions of the hydrolyzate or mixtures thereof as an active ingredient for angiotensin converting enzyme inhibition or antihypertensive composition, or abalone flesh extract of the present invention, abalone viscera Anti-obesity compositions containing extracts, abalone viscera protein extracts, hydrolysates of the protein extracts, fractions of the hydrolysates, or mixtures thereof as active ingredients are not only as pharmaceutical compositions, but also in foods and beverages. Can be used.

As a food which can add the abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the said protein extract, fraction of the said hydrolyzate, or a mixture thereof, for example, Various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

Abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate, or a mixture containing these mixtures have little toxicity and side effects, so high blood pressure, arteriosclerosis, obesity, etc. Can be used safely for long periods of time for the purpose of prevention.

Abalone flesh extract of the present invention, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate or a mixture thereof is a composition for the prevention of hypertension, arteriosclerosis, obesity, etc. Or in a beverage, wherein the amount of the composition containing the abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate or mixtures thereof in the food or beverage is total It can be added at 0.00001 to 20% by weight of the food weight, the health beverage can be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml.

As a composition for food of the present invention, in particular, a composition used as a health functional beverage is the abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fraction of the hydrolyzate as essential ingredients in the indicated ratio Or other components other than those containing a mixture is not particularly limited and may further include various flavors or natural carbohydrates and the like as a conventional beverage.

At this time, the natural carbohydrate is, for example, monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate or a mixture thereof, the food composition contains various nutrients, vitamins, minerals (electrolytes) , Flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin , Alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the food composition containing the abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fractions of the hydrolyzate, or mixtures thereof of the present invention is a natural fruit juice and fruit juice beverage and vegetable beverage It may contain flesh for preparation, and these ingredients may be used independently or in combination. In addition, the ratio of the additive is not so important, but from 0 to about per 100 parts by weight of abalone flesh extract, abalone viscera extract, abalone viscera protein extract, hydrolyzate of the protein extract, fraction of the hydrolyzate or mixture thereof It is generally selected in the range of 20 parts by weight.

Hereinafter, the present invention will be described in detail with reference to examples, but these examples are only presented to more clearly understand the present invention, and are not intended to limit the scope of the present invention. It will be determined within the scope of the technical details of the claims.

abalone

In the present invention, the war veteran ( Haliotis produced in Shin-ji, Wando-gun, Jeollanam-do) discus hannai ) was used.

Example  1 to 6 ( Comparative example  1 to 6): Abalone flesh extract

The abalone flesh was separated, washed, and then chopped and then lyophilized for 48 hours and stored at 4 ° C. for use as an abalone flesh sample, using methanol, ethanol, hexane, chloroform, ethyl acetate or butanol as an extraction solvent, respectively. 40 mL of each of these extraction solvents was added thereto, and 0.3 g of the abalone flesh sample was added thereto, followed by extraction at 150 rpm for 10 hours or more using a filter paper (Whatman No. 1). The filtrate was filtered using a rotary evaporator (EYELA, Japan). ), All the extraction solvents were evaporated to obtain an abalone flesh extract. The weight and the like of the abalone flesh extract according to the extraction solvent are shown in Table 2 below.

Extraction solvent
Extract weight (mg)
Abbreviation
Methanol
60
MM
ethanol
10
EM
Hexane
10
HM
chloroform
10
CM
Ethyl acetate
70
EAM
Butanol
20
BM

In terms of anti-obesity effect, abalone extracts obtained using the above-mentioned extraction solvents of methanol, ethanol, hexane, chloroform, ethyl acetate and butanol, respectively, are Examples 1 to 6, and angiotensin converting enzyme inhibitory effect and antihypertensive effect, respectively. In terms of, the abalone flesh extracts obtained using the above-described extraction solvents of methanol, ethanol, hexane, chloroform, ethyl acetate and butanol are Comparative Examples 1 to 6, respectively.

In Examples 1 to 6 (Comparative Examples 1 to 6), when the activity was measured in the following Experimental Example, it was used by dissolving in DMSO (Dimethyl sulfoxide) at a concentration of 10 mg / ml.

Example  7 to 12 ( Comparative example  7-12): Abalone Visceral Extract

After separating and washing the viscera of abalone, chopped, and freeze-dried for 48 hours and stored it at 4 ° C. for use as abalone viscera sample, using methanol, ethanol, hexane, chloroform, ethyl acetate or butanol as extraction solvent, respectively. 40 mL of each of these extraction solvents was added thereto, and 0.5 g of the abalone gut sample was added thereto, followed by extraction at 150 rpm for 10 hours or more using a filter paper (Whatman No. 1). The filtrate was filtered using a rotary evaporator (EYELA, Japan). ), The extraction solvent was evaporated to obtain the abalone viscera extract. The weight of the abalone viscera extract according to the extraction solvent is shown in Table 3 below.

Extraction solvent
Extract weight (mg)
Abbreviation
Methanol
190
MI
ethanol
140
EI
Hexane
120
HI
chloroform
80
CI
Ethyl acetate
78
EAI
Butanol
90
BI

In terms of anti-obesity effect, the abalone viscera extracts obtained using the above-described extraction solvents of methanol, ethanol, hexane, chloroform, ethyl acetate and butanol, respectively, are Examples 7 to 12, and angiotensin converting enzyme inhibitory effect and antihypertensive effect, respectively. In terms of, abalone viscera extracts obtained by using the extraction solvent of methanol, ethanol, hexane, chloroform, ethyl acetate and butanol, respectively, are Comparative Examples 7 to 12, respectively.

In Examples 7 to 12 (Comparative Examples 7 to 12), when the activity was measured in the following Experimental Example, it was used by dissolving at a concentration of 10 mg / ml in DMSO (dimethyl sulfoxide).

Example  13 and 14: Preparation of Abalone Visceral Protein Extract

The viscera of the abalone were separated, washed, and then lyophilized for 48 hours to prepare a powder form. 500 ml of 50 mM sodium phosphate buffer (pH 7.4) was added to 10 g of the abalone viscera powder, and extracted at 150 ° C. for 6 hours or more at 4 ° C., followed by centrifugation at 4,000 rpm to obtain a supernatant. Ammonium sulfate was added to the solution by concentration (20-50%, 50-80%) to precipitate the protein, and the protein was dialyzed at 4 ° C. for 24 hours using a dialysis tube, followed by freeze drying. Abalone visceral protein extract was obtained.

It is Example 13 that the concentration of said ammonium sulfate is 20 to 50%, and Example 14 is 50 to 80%.

In Examples 13 and 14, the activity was measured in the following Experimental Example and dissolved in DMSO (dimethyl sulfoxide) at a concentration of 10 mg / ml.

Experimental Example  One: Example  13, Example  14 and Comparative example  Determination of antihypertensive activity of 1 to 12 ( Angiotensin  Conversion enzyme inhibitory activity measurement)

Angiotensin converting enzyme (Kinase peptidyldipeptide hydrolase, EC 3.4.15.1) cleaves C-terminal dipeptides (His-Leu) of angiotensin, an inactive decapeptide, by vascular wall smooth muscle contraction. It is known to produce angiotensin, an octapeptide showing a strong blood pressure synergism, and to increase blood pressure by inactivating bradykinin, a nona peptide having a strong vasodilation effect (see FIG. 1).

In the mechanism of hypertension development, the renin-angiotensin system of FIG. 1 plays a very important role in blood pressure control. In particular, ACE is an enzyme involved in the final step of synthesizing angiotensin, an octapeptide that hydrolyzes the C-terminal dipeptide from decapepide angiotensin produced by renin. The produced angiotensin promotes the release of aldosterone in the adrenal cortex, inhibits the excretion of water and sodium, and consequently increases blood pressure by inhibiting bradykinin, a nonapeptide with vasodilation. Therefore, ACE inhibitory action has the effect of lowering blood pressure by preventing blood vessel contraction and water retention in the body.

This ACE inhibitory activity was measured according to the following method.

That is, 50 μl of 0.1 M sodium borate buffer solution (pH 8.3) and 150 μl of 2.5 mM HHL (Hippury-His-Leu) and a sample, that is, extracts of Examples 13, 14, and Comparative Examples 1 to 12 50 After the addition of ㎕ and stirred well with a vortex and pre-incubation for 10 minutes at 37 ℃. 50 μl of ACE (100 mU) was added thereto, reacted at 37 ° C. for 30 minutes, and 200 μl of 1N HCl was added to stop the reaction. As a control, distilled water was used instead of the sample. After adding 800 µl of ethyl acetate to the mixture, the mixture was stirred well and left at room temperature for 10 minutes. 600 µl of ethyl acetate, which was supernatant, was taken up in a new tube, completely dried at 120 ° C. for 1 hour, and then dissolved by adding 1 ml of 1M aqueous NaCl solution. The absorbance at 228 nm was measured using a UV-Vis spectrophotometer instrument (Ultrospec 2100 pro, GE Healthcare) and the ACE inhibitory activity was calculated as follows.

The results of measuring ACE inhibitory activity are shown in FIGS. 2 and 3 (enalapril is a conventional angiotensin converting enzyme inhibitor.)

Figure 2 is a graph showing the results of measuring the ACE inhibitory activity of the extracts of Comparative Examples 1 to 12, the abalone extract of Comparative Examples 3 to 6 (HM, CM, EAM, BM) shows the highest ACE inhibitory activity, The activity is inferior to about 30%.

Figure 3 is a graph showing the results of measuring the ACE inhibitory activity of the extracts of Examples 13 and 14, Example 13 (ammonium sulfate concentration: 20-50%) shows an ACE inhibitory activity of 45.6%, Example 14 (Ammonium sulfate concentration: 50-80%) shows an ACE inhibitory activity of 37.2%.

Experimental Example  2: Example  1 to 14 Anti-obesity  Activity measurement ( Pancreatic lipase  Inhibition activity measurement)

Fat inside the food is present in the form of triglycerides ingested fat is the action of pancreatic lipase secreted by the pancreas (Pancreatic lipase) hydrolyzed ester bonds of triglycerides and fatty acids and glycerol is formed. The degraded fatty acid and glycerol are absorbed by the mucosal cells of the small intestine and used as an energy source. After use, the remaining fatty acid and glycerol are synthesized into triacylglycerol through the monoacylglycerol pathway and accumulated in the body. Therefore, by inhibiting the activity of pancreatic lipase, which acts as an important enzyme of fat, it is possible to prevent obesity by limiting the digestion of excessively ingested fat.

Anti-obesity activity experiments were measured using the Bitou method. That is, anti-obesity activity was measured by analyzing oleic acid isolated from triolein.

90 μM triolein in 9 ml of 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pH 7.0) containing 0.1 M NaCl, gum arabic (gum arabic) 45mg, taurocholic acid (taurocholic acid) was added 9.45μM and sonicated for 5 minutes (sonication) was used as a substrate solution of the reaction solution. 15 μl of enzyme solution pancreatic lipase (1,500 U / ml), 5 μl of each sample, i.e., extracts of Examples 1 to 14, and 180 μl of the substrate solution were mixed to prepare a reaction solution for measuring lipase activity. It was reacted for 30 minutes at pH 7.0 and 37 ° C. As a control, distilled water was used instead of the sample. The oleic acid produced after the enzyme reaction was quantitatively measured. That is, after the enzyme reaction, 3 ml of a chloroform / heptane (1: 1) solution containing 2% (v / v) methanol was added to 200 µl of the reaction solution and centrifuged for 10 minutes. Separate (2,000 × g) to remove the water layer, add 1 ml of copper reaction solution and stir again for 10 minutes, and centrifuge (2,000 × g) for 10 minutes to extract the organic solvent containing oleic acid and copper salt. Take 1 ml of layer, then 0.1% (w / v) betocupro with 0.05% (w / v) 3-tert-butyl-4-hydroxyanisole After adding 1 ml of a solution of bathocuproine, absorbance was measured at 480 nm using a UV-Vis spectrophotometer device (Ultrospec 2100 pro, GE Healthcare) and pancreatic lipase activity was calculated as follows.

The results of measuring pancreatic lipase inhibitory activity are shown in FIGS. 4 and 5 (orlistat is a conventional pancreatic lipase inhibitor).

4 is a graph showing pancreatic lipase inhibitory activity of the extracts of Examples 1 to 6 and 9 to 12, wherein the abalone flesh extracts of Examples 1 to 6 are superior to the abalone viscera extracts of Examples 9 to 12; In particular, abalone flesh extract or abalone viscera extract obtained by using an extraction solvent such as hexane, chloroform, ethyl acetate, butanol and the like shows high inhibitory effect of pancreatic lipase activity.

FIG. 5 is a graph showing pancreatic lipase inhibitory activity of the extracts of Examples 13 and 14, and in the case of Example 13 (ammonium sulfate concentration: 20-50%), 34.91% of pancreatic lipase inhibitory activity, and Example 14 (sulfuric acid) Ammonium concentration: 50-80%) shows pancreatic lipase inhibitory activity of 41.31%.

Example  15-17: Abalone Visceral Protein Extract Hydrolyzate  Produce

20 ml of distilled water was added to 100 mg of the abalone viscera protein extract of Example 13, adjusted to pH 2.0 using 2 M HCl, and then 100 mM HCl (pH 2.0) solution containing pepsin (1 mg / ml), a proteolytic enzyme. Μl was added and reacted at 200 ° C. for 24 hours at 200 rpm and then incubated at 100 ° C. for 10 minutes to inactivate the proteolytic enzyme, followed by centrifugation at 8,000 rpm for 15 minutes and the supernatant. Was taken and lyophilized to obtain a hydrolyzate (Example 15) of the abalone viscera protein extract of the present invention.

20 ml of distilled water was added to 100 mg of the abalone viscera protein extract of Example 13, and 100 µl of a 50 mM sodium phosphate (pH 7.0) buffer solution containing chymotrypsin (1 mg / ml), a proteolytic enzyme, was added thereto. After the reaction at 200 rpm for 24 hours, the proteolytic enzyme was inactivated by agitation at 100 ° C. for 10 minutes, and then centrifuged for 15 minutes at a speed of 8,000 rpm, the supernatant was taken, and lyophilized. Hydrolysates of Abalone gut protein extract (Example 16) were obtained.

20 ml of distilled water was added to 100 mg of abalone viscera protein extract of Example 13, adjusted to pH 8.0 using a 50 mM sodium phosphate buffer solution, and 50 mM sodium phosphate (pancreatin) (1 mg / ml) containing proteolytic enzyme. pH 8.0) 100 μl of the buffer solution was added, the reaction was carried out at a temperature of 37 ° C. for 24 hours at 200 rpm, followed by agitation at 100 ° C. for 10 minutes to inactivate the proteolytic enzyme, and then at a speed of 8,000 rpm 15. Centrifuged for a minute, the supernatant was taken and lyophilized to obtain a hydrolyzate (Example 17) of the abalone viscera protein extract of the present invention.

The optimum reaction temperature and pH of each hydrolase in the preparation of the hydrolyzate of the abalone viscera protein extracts of Examples 15 to 17 are shown in Table 1 above.

When measuring the activity in the following Experimental Examples for Examples 15 to 17 was used dissolved in a concentration of 10mg / ml in DMSO (dimethyl sulfoxide).

Experimental Example  3: Example  Determination of antihypertensive activity of 15 to 17 Angiotensin  Conversion enzyme inhibitory activity measurement)

Antihypertensive activity (angiotensin converting enzyme inhibitory activity) was measured by the same method as in Example 1 except that the hydrolyzate of Examples 15 to 17 was used instead of the sample solution, that is, the extract of Example 13, etc. It was.

The results are shown in Figure 6, the hydrolyzate of Examples 15 to 17 showed excellent ACE inhibitory activity as a whole, especially in the case of Example 15 treated with pepsin shows very significant ACE inhibitory activity.

Example  18: Abalone Visceral Protein Extract Hydrolyzate Fraction  Produce

Large-scale preparative chromatography (prep-LC, JAI, Japan) was used to obtain a fraction of the hydrolyzate of the abalone viscera protein extract. Separation conditions are shown in Table 4 below.

device
JAI LC-9104
column
GS-310 (20 × 500mm)
Mobile phase
water
Flow rate
3.5ml / min
Detector
UV 220nm

The hydrolyzate of the abalone viscera protein extract of Example 15 was separated through a GS-310 column (20 × 500 mm) using a prep-LC system. Elution was carried out at a flow rate of 3.5 ml / min (JAI LC-9104, JAPAN) and the absorbance was measured at 220 nm using a device to identify the three major peaks, the fractions (A, B, C) were collected and lyophilized A fraction of the hydrolyzate of the abalone gut protein extract was obtained (see FIG. 7).

When measuring the activity in the following Experimental Example for this Example 18 was used dissolved in a concentration of 10mg / ml in DMSO (dimethyl sulfoxide).

Experimental Example  4: Example  18 antihypertensive activity measurements Angiotensin  Conversion enzyme inhibitory activity measurement)

Antihypertensive activity (angiotensin converting enzyme inhibitory activity) was measured in the same manner as in the measurement method of Experimental Example 1, except that the fraction of Example 18 was used instead of the sample solution, that is, the extract of Example 13.

The results are shown in Figure 7, the fraction of Example 18 showed a good overall ACE inhibitory activity, in particular, the second fraction of the three fractions B fraction can be confirmed that the very significant ACE inhibitory activity.

Claims (7)

An antihypertensive pharmaceutical composition comprising an abalone viscera protein extract, a hydrolyzate of the protein extract, and a mixture thereof, as an active ingredient. An antihypertensive pharmaceutical composition comprising a fraction of the hydrolyzate of the abalone viscera protein extract as an active ingredient, wherein the fraction is obtained by performing large-scale preparative chromatography using water as an eluting solvent. An anti-obesity pharmaceutical composition comprising an abalone flesh extract, abalone viscera extract, abalone viscera protein extract, a hydrolyzate of the protein extract and a mixture thereof as an active ingredient. An anti-obesity pharmaceutical composition comprising a fraction of the hydrolyzate of the abalone viscera protein extract as an active ingredient, the fraction obtained by performing large-scale preparative chromatography using water as the elution solvent. The hydrolyzate according to any one of claims 1 to 4, wherein the hydrolyzate comprises pepsin, trysin, chymotrypsin, pancreatin, alkalase ( alcalase), flavozyme (flavourzyme), papain (papain) and a mixture obtained by treatment with a proteolytic enzyme selected from the group consisting of. Abalone visceral protein extract, a hydrolyzate of the protein extract and a mixture thereof, containing the selected from the group consisting of an active ingredient, preventing hypertension or obesity food composition. A fraction containing the hydrolyzate of the abalone viscera protein extract as an active ingredient, the fraction obtained by performing large-scale preparative chromatography using water as the elution solvent, food composition for preventing hypertension or obesity.

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